Multidrug resistant Clostridium difficile ribotype 027 in southwestern Virginia, 2007 to 2013.

The excess from fecal samples submitted to a centralized laboratory in Roanoke, Virginia for routine C. difficile testing was used for this research study. We tested all samples, including any formed samples usually not assayed in diagnostic laboratories. Our first aim was to rank ribotypes by their frequency. Between 2007 and 2013, fluoroquinolone resistant 027 (027FQR), a multi-drug resistant ribotype, was 32% of 3118 Clostridium difficile isolates and the most common of 128 ribotypes. 027FQR was in 45% of cytotoxin positive but only 17% of cytotoxin negative fecal samples (p = 0.001) and 34% of unformed but only 21% of formed stool samples (p = 0.001), strong associations with features of symptomatic infection. Conversely, 014/020 (10% of isolates, third most common ribotype) was more often in unformed than formed stools (14% versus 9%; p = 0.002) and in cytotoxin negative than cytotoxin positive samples (11% versus 8%, p = 0.01). Fecal lactoferrin levels, an indication of intestinal inflammation, were significantly higher with 027FQR than with 014/020 infections (median 308 versus 26 ng/mL, p = 0.02). 027FQR fecal bioburdens and toxin levels were significantly higher than their 014/020 equivalents (median 104.1 versus 103.2/g feces, p = 0.01; median TcdA 58.7 versus 1.3 ng/g feces, p = 0.04; median TcdB 43.4 versus 0.3 ng/g feces, p = 0.001). Binary toxin was present in 40% of 027FQR positive samples but none of the 014/020 or non-toxigenic C. difficile positive samples. 027FQR made no more TcdA/cell than did 014/020 (p = 0.7) but did make close to significantly more TcdB/cell (p = 0.08).

Clostridium difficile ribotype 027 is most prevalent among inpatients admitted from long-term care facilities.

Intestinal inflammation was evaluated using faecal lactoferrin and ribotype in 196 hospitalized adults with Clostridium difficile infection to determine the impact of ribotype 027 in long-term care facilities (LTCFs). LTCF residents (n=28) had greater antibiotic use (P=0.049) and more ribotype 027 infection [odds ratio (OR): 4.87; 95% confidence interval (CI): 2.02-11.74; P<0.01], compared to those admitted from home. Patients infected with ribotype 027 strains had worse six-month mortality (OR: 1.90; 95% CI: 1.08-3.34; P=0.03) and more inflammation (95.26 vs 36.08 µg/mL; P=0.006), compared to those infected with non-027 strains. This study was not designed to determine acquisition site, but, in this population, suggests that the location from which the patient has been admitted is strongly associated with ribotype 027 and more severe C. difficile disease.

Ribotype 027 Clostridium difficile infections with measurable stool toxin have increased lactoferrin and are associated with a higher mortality.

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n = 191) were positive by toxigenic culture and 61% (n = 129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 µg/g versus 106 µg/g stool; p < 0.0001). Forty percent of patients (n = 85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79% vs. 50%; p < 0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 µg/g; p = 0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18%; p = 0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR = 1.1), intensive care unit treatment (OR = 2.7), CI (OR = 1.2), 027 CDI (OR = 2.1), white blood cell count (OR = 1.0), albumin level (OR = 0.1), and stool toxin-positive 027 CDI (OR = 2.5) were significantly associated with 100-day mortality (p < 0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.

Elevated lactoferrin is associated with moderate to severe Clostridium difficile disease, stool toxin, and 027 infection.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes.

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Clostridium difficile prevalence rates in a large healthcare system stratified according to patient population, age, gender, and specimen consistency.

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients<20 years of age (23%). We identified 31 different toxigenic ribotypes from a sampling of 190 isolates that showed the lowest diversity in NH. Fluoroquinolone resistance was observed in 93% of the 027 isolates, all of the 053 isolates, and in four other ribotypes. We observed different rates for toxigenic C. difficile in stratified patient populations, with the highest rate for NH, a low overall nontoxigenic rate, and fluoroquinolone resistance.

Staphylococcus aureus: the toxic presence of a pathogen extraordinaire.

Staphylococcus aureus is a facultative, Gram-positive coccus well known for its disease-causing capabilities. In particular, methicillin and vancomycin resistant strains of S. aureus (MRSA and VRSA, respectively) isolated globally represent daunting medical challenges for the 21(st) Century. This bacterium causes numerous illnesses in humans such as food poisoning, skin infections, osteomyelitis, endocarditis, pneumonia, enterocolitis, toxic shock, and autoimmune disorders. A few of the many virulence factors attributed to S. aureus include antibiotic resistance, capsule, coagulase, lipase, hyaluronidase, protein A, fibronectin-binding protein, and multiple toxins with diverse activities. One family of protein toxins is the staphylococcal enterotoxins (SEs) and related toxic shock syndrome toxin-1 (TSST-1) that act as superantigens. There are more than twenty different SEs described to date with varying amino acid sequences, common conformations, and similar biological effects. By definition, very low (picomolar) concentrations of these superantigenic toxins activate specific T-cell subsets after binding to major histocompatibility complex class II. Activated T-cells vigorously proliferate and release proinflammatory cytokines plus chemokines that can elicit fever, hypotension, and other ailments which include a potentially lethal shock. In vitro and in vivo models are available for studying the SEs and TSST-1, thus providing important tools for understanding modes of action and subsequently countering these toxins via experimental vaccines or therapeutics. This review succinctly presents the pathogenic ways of S. aureus, with a toxic twist. There will be a particular focus upon the biological and biochemical properties of, plus current neutralization strategies targeting, staphylcoccocal superantigens like the SEs and TSST-1.

Multicenter evaluation of a new screening test that detects Clostridium difficile in fecal specimens.

Clostridium difficile causes approximately 25% of nosocomial antibiotic-associated diarrheas and most cases of pseudomembranous colitis. We evaluated C. DIFF CHEK, a new screening test that detects glutamate dehydrogenase of C. difficile. Our results showed that this test was comparable to PCR in sensitivity and specificity and outperformed bacterial culture.

Effects of low levels of ciprofloxacin on a chemostat model of the human colonic microflora.

To study the utility of an in vitro model system for assessing the effect of low concentrations of a fluoroquinolone (FQ) drug on the ecology of the human intestinal microflora, chemostats containing human fecal flora were exposed to 0.43, 4.3, and 43microg of ciprofloxacin (CI) per milliliter. Prior to and during drug exposure, we assayed short-chain fatty acids (SCFA), bacterial populations, and the relative levels of susceptibility of these populations to CI and trovafloxacin (TV), a newer related FQ with increased activity against anaerobes. The degree to which CI affected the chemostat ecology was measured statistically by comparing observed data with the corresponding predicted "no effect" level. No changes in total SCFA were observed; only butyrate was significantly higher at the intermediate and high-dose levels. Enterococci counts and the levels of susceptibility to CI among enterococci were also unaffected. Escherichia coli counts decreased in a dose-dependent manner. Susceptibility levels in E. coli followed no interpretable pattern. Bacteroides fragilis group (BfG) counts decreased significantly following exposure to 43 and 4.3microg/mL CI. Ciprofloxacin susceptibility among the BfG in these chemostats was not determined because the BfG counts were too low (less than 30 colonies per plate) when undiluted chemostat samples were plated. However, within 2 days of exposure to 0.43microg/mL CI, the percentage of BfG resistant to 4microg/mL CI increased to over 95%. Before exposure, all BfG were susceptible to both CI (2microg/mL) and TV (0.25microg/mL). All BfG isolated during exposure were resistant to both CI (4microg/mL) and TV (2microg/mL). Resistance selection in the BfG was unexpected as the MIC(90) of CI for B. fragilis is 8microg/mL. Since the average colon flora is about 20% B. fragilis and other bacteroides, CI may impact the human gut flora even at subtherapeutic levels.

Genotyping of enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-associated diarrhea and food poisoning in North America.

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.

Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops.

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.

Enhanced performance of elemental copper-vapor lasers by use of H(2)-HCl-Ne buffer-gas mixtures.

The output power from a 25-mm-diameter (volume, 0.49 L) and a 40-mm-diameter (volume, 1.9 L) copper-vapor laser (nominally 20- and 65-W devices, respectively) was approximately doubled to >50 and >100 W , respectively, by addition of small partial pressures of both H(2) and HCl to the neon buffer gas. The specific output powers of these lasers are believed to be records for any copper-vapor lasers of this size.

Kinetics modeling of a pulsed Cu-Ne discharge: potential for new ultraviolet laser transitions in Cu II.

A rate-equation analysis has been used to investigate the feasibility of exciting new UV laser transitions in Cu II (3d(9)4p-3d(9)4s) by use of a pulsed Cu-Ne discharge. The model predicts average output powers in excess of 100 mW at 10 kHz from the combined output at 201.5 and 211.2 nm.

Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus".

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.

Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III.

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.

Assignment of the agent of Tyzzer's disease to Clostridium piliforme comb. nov. on the basis of 16S rRNA sequence analysis.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.